Systemic lupus erythematosus (SLE) is a chronic debilitating disease that is characterized by high titers of autoantibodies with specificity for nuclear autoantigens. It is considered to arise from interactions between underlying genetic susceptibility and environmental factors, with different combinations resulting in inter- individual variations in disease manifestations and therapeutic responsiveness. Directed therapeutic targeting of specific pathways has not proven effective, which suggests that the current understanding of pathogenesis is incomplete. The goal of the proposed project is to test the hypothesis that the survival of immature autoreactive B cells, the preferential development of antinuclear autoreactive B cells and the status of mature autoreactive B cells (immunocompetent vs. anergic/tolerogenic) is decided during the transitional stage 1 (T1) of development and requires constitutive T1 B cell expression of interferon ? (IFN?). This paradigm shifting hypothesis is based on data generated using a combination of mixed-bone marrow chimeras, flow cytometry with tetramer or idiotype antibody selection, and high-throughput single-cell analysis to interrogate type I interferon (IFN) networks in B cell from patients with SLE and lupus-prone BXD2 mice. The data suggest that the currently known molecular and cellular aberrations are preceded by a single primary pathogenic event, i.e., production of IFN? by T1 B cells. The data suggest that the survival of the very early T1 cells is dependent on endogenous expression of IFN?. This subset of T1 B cells express endogenous IFN?, which leads to their development into T1 B cells that produce IFN?. The ability of T1 B cells to escape negative B-cell receptor (BCR)-mediated selection is dictated by the type I IFN-determined responsiveness of this T1 subset to BCR-mediated signaling in combination with stimulation of TLR signaling (TLR7 or TLR9) by apoptotic debris. This preferentially permits escape of nucleic antigen-autoreactive B cells. We have further identified that the T1 B cell compartment contains distinct subsets of cells. As these included subsets with transcriptional profiles that parallel the phenotypes of mature and anergic suppressive regulatory (Breg) cells and their immunogenic counterparts that predominate in SLE, our new data suggest that this phenotypic switch in the mature B cells is imprinted during the T1 stage. These data also suggest that development of type I IFN network associated T1 B cells in BXD2 mice is associated with type I IFN-inducing transcription factors (IRF3 and IRF7) whereas induction of the Foxp3+ regulatory T1 B cells is associated with transcription factor, ID3. Notably, these data not only suggest suppression of precursors of T1-Breg is a novel pathogenic framework in SLE but also provide the tool, i.e., the gene expression signatures in sorted transitional B cells, that enable its analysis. We will test the critical elements of our overall hypothesis through three Specific Aims that will: (1) Distinguish the roles of the constitutive IFN? signaling and the microenvironmental signals in driving the development of T1 B cells; (2) Identify if the initial T1 B cell transcriptional imprinting is maintained at the T2/MZP-MZ-FO stage of development; and (3) Determine if T1 B-cell endogenous IFN? promotes a type I IFN network that suppresses the precursors of regulatory B cells at the T1 stage in SLE patients. The scientific premises of the proposed work include the parallels between transcriptional profiles in the T1 B cells and mature B cells and the segregation of type I IFN associated vs Breg associated transcriptional profiles in the T1 B cells. We have acquired reporter mouse strains for IFN?, IL-10, Foxp3 and Id3 to interrogate discreet developmental stages within and derived from transitional T1 B subsets. The information gained will have a broad-based impact on the field and improve the care of VA patients with SLE by identifying strategies to improve the efficacy of targeted biologic therapies together with non- invasive precision medicine guided-approaches.